The aim of this project is to investigate the roles of DNA repair and DNA replication in predicting toxicity (cell killing and neoplastic transformation) in repair-deficient and repair-proficient 10T1/2 cells. We have found that CH3 10T1/2 cells can be made DNA repair-deficient by depriving them of isoleucine. Compared to repair-proficient 10T1/2 cells (isoleucine-sufficient), repair-deficient cells are more sensitive to the cell killing and transforming effects of directly acting alkylating agents, such as methyl-N'-nitro-N-nitrosoguanidine. In this project we will determine whether the DNA repair deficiency involves excision or post-replication repair (or both) and, by utilizing synchronized cells, we will examine the role of DNA replication in causing toxicity. The competence of DNA excision repair will be evaluated in G1 phase cells by measuring single strand breakage and gap-formation, removal of alkylated bases, repair DNA synthesis, and ligation. Post-replication repair will be evaluated in S phase cells by determining the rate of formation and size of primary pieces and by measuring the accumulation of gaps in DNA. Toxicity is assessed by determining the colony-forming efficiency of treated and control cells.